Method for producing preparations for electromicroscopic examination

ABSTRACT

A method for the production of preparation for electron microscopic investigation which includes irradiating the objects to be investigated in a high vacuum with electrons of a low energy and regulating an accelerating voltage to control the irradiation-chemical reaction.

United States Patent 1 Grasenick et al.

METHOD FOR PRODUCING PREPARATIONS FOR ELEOTROMICROSCOPIC EXAMINATXON Inventors: Fritz Grasenick; Erich Jakopic, both of Steyrergasse 17, A 8010 Graz, Austria Filed: Dec. 7, 1970 App]. No.: 95,725

Foreign Application Priority Data Sept. 24, 1969 Austria ..9020/69 US. Cl. ..250 /49.5 TE, 250/83 CD, l56/2 Int. Cl ..H0lj 37/00. Field of Search 156/2; 250/83 CD Apr. 24. W73

[56] References Cited UNITED STATES PATENTS 3 6l2,87l lO/l97l Crawford et al. ..25()/83 CD Primary ExaminerJ. Steinberg Attorney-Watson, Cole, Grindle& Watson 57 ABSTRACT A method for the production of preparation for electron microscopic investigation which includes irradiating the objects to be investigated in a high vacuum with electrons of a low energy and regulating an accelerating voltage to control the irradiation-chemical reaction.

i 3 Claims, 2 Drawing Figures METHOD FOR PRODUCING PREPARATIONS FOR ELECTROMICROSCOPIC EXAMINATION This invention relates to a process for the production of preparations for electron microscopic investigation.

As is well known, the irradiation of organic high polymers with particles or quanta by radiation-chemical reaction frequently leads to a linking of the substances. This linking begins above a certain dose of irradiation (gel dose) and leads to the formation of an insoluble share which quickly increases with an increasing dose and which approaches a border value asymptotically. The development of an insoluble share in the irradiated substancemakes it appear possible to make use of this effect for the production of electron microscopic preparations of certain organic materials, such as for example polyethylene, rubber, and others. The question frequently arises indeed as to the structure of inhomogeneous organic materials which the expert working with the electron microscope tries to answer with the production and inventigation of thin slices, imprints and sheathings of the pertinent substance. However, the methods mentioned have various drawbacks such as for example, the development of deformations in the case of cutting thin slices or the effects which occur perhaps in the case of penetration of embedding agents into the substance. Impressions and sheathings mainly represent only the microgeometry of the surface and they will either permit no insight at all or only a very limited insight into the inside of the substance. Also the process of lifting thin layers from the organic substances by etching by means of activated oxygen and application of a layer of gelatin often does not lead to the goal, especially not when dealing with porous material. In these and other cases, the process according to the present invention will offer a remedy.

It is an object of the invention to create a process for the production of preparations for the electron microscopic investigation which no longer shows the previously explained defects of the known processes.

According to this invention, it will be solved in the case of a process for the production of preparations for the electron microscopic investigation by making use of radiation-chemical reactions by the fact that the objects to be investigated are irradiated in a high vacuum with electrons of low energy of maximum keV, so that within the area of a desirable penetration depth by the electrons which can be regulated by adjustment of accelerating voltage, a radiation-chemical reaction for example a linking of the material of the object will be brought about after which the irradiated layer having a different solubility is exposed by treatment of the nonirradiated object material and thus is ready for the known electron microscopic trans-irradiation.

The basis of the process according to the present invention is therefore the irradiation with electrons of a relatively low energy of the material that is to be investigated and to be sure under conditions which will largely prevent an impermissible heating of the preparation and a growth of disturbing layers of polymerize (contamination). Because of their low energy the depth of penetration by the electrons is slight. The thickness of this layer can be estimated according to known formulas, whereby one will obtain numerical values, for example, for polyethylene:

E(kev d(A) (E energy of the electrons in keV d depth of penetration in Angstrom units) Since thicknesses of layers of only a few hundred to a maximum of a few thousand A are needed for the electron microscopic investigation in the case of the customary irradiation voltages of 40 100 kV (depending on the density and a possibly existing porosity of the substance which is being irradiated), one must therefore use the irradiation, electron energy of a few tenths up to a few keV. The values given in the table however must be considered only as guide values and in the case of depth of penetration mentioned one is dealing with the so-called practical range, that is the distance from the surface of the preparation after passing through so that the kinetic energy of the electrons in the agent disappears. Nevertheless it becomes clear from the experiments made that the energy range used supplied fitting thicknesses of the layer.

For the linking of high polymer, a certain minimum irradiation dose is required.

Further object of the invention will be apparent from the following description when considered in connection with the accompanying drawings in which:

FIG. 1 is a diagram showing the increase with the dose of the insoluble share of branched polyethylene, and

FIG. 2 is a diagrammatic view of a ray producer system with a cooling means and circuit.

One can recognize from the curve of FIG. 1 that above a dose of approximately megarad it does not substantially increase any more.

For the production of natural or general layers according to the invention, an electron ray producing system will be required in which the beam current and the beam voltage can be regulated as much as possible independently of one another and in which an even density of the beam current on the preparation will be produced. Experiments with the ray producing system of a TV picture tube were carried out, whose oxide cathode was replaced by a large surface cathode coated with lanthanum boride. Lanthanum boride is suitable as an emission layer in the case of good emission characteristics, above all, whenever the recipient is being frequently ventilated because an action by the air will not harm this cathode in its cold state. FIG. 2 shows the diagram of the ray producer system with a cooling chamber and also the circuit for operation of the ray producing system. By regulation of the Wehneit voltage and of the focusing voltage, the density of the beam current on the preparation can be adjusted and the accelerating voltage of the electrodes can be varied dependent thereon. The numeral 1 on the drawing designates the electron gun, 2 the electron lens, 3 the cooling chamber, 4 a thermoelement, 5 the Faraday cage, 6 an insulating ring, 7 a plate for the preparation and 8 the preparation itself. Further letters A, B and C designate sources for the voltage and D a recorder.

1f the irradiation of the preparation in the high vacuum (p 10 torr) is made without any special measures, then on the surface of the preparation a considerable or violent separation of layers of polymerizate (contamination) will occur. These layers originate as is known from hydrocarbon vapors which are present in standard installations even in the case of refined methods of operation. The vacuum pumps and gaskets, but also other components, which are located in the vacuum chamber and to which remnants of organic substances cling, are the main sources of these hydrocarbon vapors. These hydrocarbon vapors strike every surface inside the vacuum chamber with a finite dwell time and then are converted to a solid film by radiation-chemical reactions brought forth by the striking electrons. Now this disturbing phenomenon can be prevented for the most part by lowering the partial pressure of the hydrocarbon in the neighborhood of the preparation by the arrangement of a chamber cooled with liquid nitrogen. In that case the temperature of the cooling chamber should remain below l 30 C. At the same time, however, the object is to be at room temperature. FIG. 2 shows on the left-hand bottom, in section, the diagram of the cooling chamber. For the measurement of the preparation current, a Faraday cage mounted on the inside, is inserted into the cooling chamber.

For the production of the natural layers according to the invention, foils made of polyvinyl alcohol, polyethylene terephthalate (Mylar), acetobutyrate (Triafol), also millipore filters and nucleopore filters were irradiated. Investigations were made with Mylar foils, millipore filters and nucleopore filters.

Prior to the irradiation of the Mylar foils, the foil was cauterized or etched with activated oxygen in a high frequency gas discharge for the purpose of developing the structure. One can easily recognize the etching structure on the irradiated materials which shows itself in the natural layer. Besides the surface geometry, one can make visible here, inhomogeneous characteristics going beyond an imprint, remnants of catalysts, foreign substances, etc.

in the case of the investigation of millipore filters, a foam-like structure of the filter shows that the depth of penetration of the electrons is considerable since the thickness of the masses is light because of the porosity.

In this case the advantage of the natural layer process becomes particularly effective.

In pictures of nucleopore filters, one can recognize the homogeneousness of the pores which continue to the inside of the filter as approximately cylindrical channels, and these filters are built up differently than the foam-like diaphragm filters and they are particularly interesting in the cases of analytical work. The irradiated material shows that the channels follow some holes as hose-like structures, but in most cases however these formations are lacking since they are torn away during the separation process. It is probable that by a high accelerating voltage and because of the greater depth of penetration brought forth thereby, a better mechanical stability of the hose-like formations will be achieved. Probably, in this case too as in similar cases, the application of a reinforcing layer by evaporating quartz or gold prior to dissolving the organic substance and removing it will be advantageous for an increase in the stability and for the achievement of a cohesive pre aration.

he faithfulness in rendering pictures obtained with this process according to the invention is connected naturally with the type and magnitude of the effects of the induced rays which are caused by the electrons, and furthermore with the interference of the structure caused by the separation process. The linking of the frame of the organic substance brought about by the irradiation which frame consists mostly of carbon after a sufficiently high dose of rays (depending on the substance) is connected with a loss in mass. It is caused in the first place by the loss of hydrogen but depending on the compounding of the material, other gases too are liberated, such as for example C0, C0 CH N etc. The loss in mass may amount to 50 percent and more.

The separation process according to the invention is particularly suited also for the investigation of biological objects as well as complex colloidal systems, preferably in connection with freeze drying or low temperature sublimation (for example, in the case of swol len plastics or plastic emulsions), since a presentation of a third dimension becomes possible therewith. Furthermore, in contrast to the imprint technique, the differences in the thickness of the mass (enclosures, pores, etc.) can be made directly visible.

We claim: 1. A process for the preparation of organic polymers for microscopic examination, comprising the steps of:

mounting an organic polymer specimen within a vacuum chamber, said organic polymer being selected from the group consisting of polyvinyl alcohol, polyethylene terephthalate, and acetobutyrate; reducing the pressure within said chamber to at least 10' torr.;

cooling the portions of said chamber remote from said specimen to at least l30 C. to reduce the partial pressures of hydrocarbons within said chamber and maintaining the specimen at substantially ambient temperature;

irradiating said specimen with a beam of electrons at an even beam density and the voltage of said electron beam is in the range of several tenths of a volt to several keV; and

removing at least a portion of the non-irradiated organic polymer.

2. A process as in claim 1 wherein the organic polymer is either branched or linear polyethylene and said irradiation dosage is in the range of 20 to Mrad.

3. A process as in claim 1 further comprising a step of etching said organic polymer with activated oxygen in a high frequency gas discharge prior to irradiating with said electron beam. 

1. A process for the preparation of organic polymers for microscopic examination, comprising the steps of: mounting an organic polymer specimen within a vacuum chamber, said organic polymer being selected from the group consisting of polyvinyl alcohol, polyethylene terephthalate, and acetobutyrate; reducing the pressure within said chamber to at least 10 5 torr.; cooling the portions of said chamber remote from said specimen to at least -130* C. to reduce the partial pressures of hydrocarbons within said chamber and maintaining the specimen at substantially ambient temperature; irradiating said specimen with a beam of electrons at an even beam density and the voltage of said electron beam is in the range of several tenths of a volt to several keV; and removing at least a portion of the non-irradiated organic polymer.
 2. A process as in claim 1 wherein the organic polymer is either branched or linear polyethylene and said irradiation dosage is in the range of 20 to 80 Mrad.
 3. A process as in claim 1 further comprising a step of etching said organic polymer with activated oxygen in a high frequency gas discharge prior to irradiating with said electron beam. 